Polynucleotides encoding a transcriptional response regulator of Streptococcus pneumoniae

ABSTRACT

The invention provides Response regulator polypeptides and DNA (RNA) encoding Response regulator polypetides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing Response regulator polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims benefit of GB application number 9609021.2,filed May 1, 1996.

RELATED APPLICATIONS

This application claims benefit of GB application number 9609021.2,filed May 1, 1996.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the Response regulator family, hereinafter referred toas "Response regulator".

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This has created a demandfor both new anti-microbial agents and diagnostic tests for thisorganism.

While certain Streptococcal factors associated with pathogenicity havebeen identified, e.g., capsule polysaccharides, peptidoglycans,pneumolysins, PspA Complement factor H binding component, autolysin,neuraminidase, peptide permeases, hydrogen peroxide, IgA1 protease, thelist is certainly not complete. Further very little is known concerningthe temporal expression of such genes during infection and diseaseprogression in a mammalian host. Discovering the sets of genes thebacterium is likely to be expressing at the different stages ofinfection, particularly when an infection is established, providescritical information for the screening and characterization of novelantibacterials which can interrupt pathogenesis. In addition toproviding a fuller understanding of known proteins, such an approachwill identify previously unrecognised targets.

Many two component signal transduction systems (TCSTS) have beenidentified in bacteria (Stock, J. B., Ninfa, A. J. & Stock, A. M.(1989)Microbiol. Rev. 53, 450-490). These are involved in the bacterium'sability to monitor its surroundings and adapt to changes in itsenvironment. Several of these bacterial TCSTS are involved in virulenceand bacterial pathogenesis within the host.

Response regulators are components of the TCSTS. These proteins arephosphorylated by histidine kinases and in turn once phosphorylatedeffect the response, often through a DNA binding domain becomingactivated. The response regulators are characterized by a conservedN-terminal domain of approximately 100 amino acids. The N-terminaldomains of response regulators as well as retaining five functionallyimportant residues, corresponding to the residues D12, D13, D57, T87,K109 in CheY (Matsumura, P., Rydel, J. J., Linzmeier, R. & Vacante, D.(1984) J. Bacteriol. 160, 36-41), have conserved structural features(Volz, K. (1993) Biochemistry 32, 11741-11753). The 3-dimensionalstructures of CheY from Salmonella typhimurium (Stock, A. M., Mottonen,J. M., Stock, J. B. & Schutt, C. E. (1989) Nature, 337, 745-749) andEscherichia coli (Volz, K. & Matsumura, P. (1991) J. Biol. Chem. 266,15511-15519) and the N-terminal domain of nitrogen regulatory protein Cfrom S. typhimurium (Volkman, B. F., Nohaile, M. J., Amy, N. K., Kustu,S. & Wemmer, D. E. (1995) Biochemistry, 34 1413-1424), are available, aswell as the secondary structure of SpoOF from Bacillus subtilis (Feher,V. A., Zapf, J. W., Hoch, J. A., Dahlquist, F. W., Whiteley, J. M. &Cavanagh, J. (1995) Protein Science, 4, 1801-1814). These structureshave a (a/b)5 fold. Several structural residues are conserved betweendifferent response regulator sequences, specifically hydrophobicresidues within the β-sheet hydrophobic core and sites from thea-helices. This family of response regulators includes YYCF protein fromBacillus subtilis. YYCF is a hypothetical response regulator found inthe RocR-PurA intergenic region of B. subtilis chromosome (Ogasawara,N., Nakai, S. & H. Yoshikawa, (1994) DNA Res 1:1-14 ).

Histidine kinases are components of the TCSTS which autophosphorylate ahistidine residue. The phosphate group is then transferred to thecognate response regulator. The Histidine kinases have five shortconserved amino acid sequences (Stock, J. B., Ninfa, A. J. & Stock, A.M.(1989) Microbiol. Rev. 53, 450-490, Swanson, R. V., Alex, L. A. &Simon, M. I.(1994) TIBS 19 485-491). These are the histidine residue,which is phosphorylated, followed after approximately 100 residues by aconserved asparagine residue. After another 15 to 45 residues a DXGXGmotif is found, followed by a FXXF motif after another 10-20 residues.10-20 residues further on another glycine motif, GXG is found. The twoglycine motifs are thought to be involved in nucleotide binding.

Among the processes regulated by TCSTS are production of virulencefactors, motility, antibiotic resistance and cell replication.Inhibitors of TCSTS proteins would prevent the bacterium fromestablishing and maintaining infection of the host by preventing it fromproducing the necessary factors for pathogenesis and thereby haveutility in anti-bacterial therapy.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown Bacillus subtilis YYCF protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel Response regulator polypeptides by homology betweenthe amino acid sequence set out in Table 1 SEQ ID NO:2! and a knownamino acid sequence or sequences of other proteins such as Bacillussubtilis YYCF protein.

It is a further object of the invention to provide polynucleotides thatencode Response regulator polypeptides, particularly polynucleotidesthat encode the polypeptide herein designated Response regulator.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding Response regulatorpolypeptides comprising the sequence set out in Table 1 SEQ ID NO:1!which includes a full length gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel Response regulator protein from Streptococcus pneumoniaecomprising the amino acid sequence of Table 1 SEQ ID NO:2!, or a variantthereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Streptococcus pneumoniae 0100993 strain contained in thedeposited strain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding Response regulator, particularly Streptococcuspneumoniae Response regulator, including mRNAs, cDNAs, genomic DNAs.Further embodiments of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulvariants thereof, and compositions comprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of Response regulator and polypeptidesencoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStreptococcus pneumoniae referred to herein as Response regulator aswell as biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of Response regulator polypeptide encoded by naturallyoccurring alleles of the Response regulator gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned Response regulator polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing Responseregulator expression, treating disease, for example, otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, assaying genetic variation,and administering a Response regulator polypeptide or polynucleotide toan organism to raise an immunological response against a bacteria,especially a Streptococcus pneumoniae bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to Response regulator polynucleotide sequences, particularlyunder stringent conditions.

In certain preferred embodiments of the invention there are providedantibodies against Response regulator polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided Response regulator agonists and antagonists, preferablybacteriostatic or bacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a Response regulator polynucleotide or a Response regulatorpolypeptide for administration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% "identity" to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel Response regulator polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novelResponse regulator of Streptococcus pneumoniae, which is related byamino acid sequence homology to Bacillus subtilis YYCF polypeptide. Theinvention relates especially to Response regulator having the nucleotideand amino acid sequences set out in Table 1 SEQ ID NO:1! and Table 1 SEQID NO:2! respectively, and to the Response regulator nucleotidesequences of the DNA in the deposited strain and amino acid sequencesencoded thereby.

                                      TABLE 1                                     __________________________________________________________________________    Response regulator Polynucleotide and Polypeptide Sequences                   __________________________________________________________________________    (A) Sequences from Streptococcus pneumoniae Response regulator                polynucleotide                                                                sequence  SEQ ID NO: 1!.                                                      5'-1  ATCGAATTAT                                                                            TGGTAATATT                                                                            GGTGATTTCA                                                                            GACCCACCTT                                                                            GCATATCAAG                              51    AACCTTGGAA                                                                            TAATCTCCTG                                                                            CAATCAAATC                                                                            TTTAACCTTT                                                                            TGATTGATTT                              101   GCTTCAACTG                                                                            AATATTATCA                                                                            CGTCTATTTC                                                                            CCAGTAATAA                                                                            GAGGGTCACA                              151   ACAAGGATGA                                                                            AACCTAACAA                                                                            AATCAGGATA                                                                            AAGATAAAAT                                                                            CTCTGGTAAA                              201   AATGGTTTGT                                                                            TTCAGTAAAT                                                                            CAAGCATTAT                                                                            TTCTCATGTA                                                                            ATACCCTACA                              251   CCACGGCGCG                                                                            TCAAGATATA                                                                            CTCTGGTCGG                                                                            CTGGGCGTAT                                                                            CTTCAATCTT                              301   CTCACGCAGA                                                                            CGTCGTACAG                                                                            TCACATCAAC                                                                            TGTACGGACA                                                                            TCACCAAAAT                              351   AGTCATAACC                                                                            CCAGACAGTC                                                                            TCAAGCAAGT                                                                            GTTCGCGCGT                                                                            GATGACTTGA                              401   CCTGTATGCG                                                                            ATGCTAAATG                                                                            ATACAAAAGC                                                                            TCAAATTCAC                                                                            GATGGGTTAA                              451   GTCTAGTTCT                                                                            TCGCCATATT                                                                            TTTTAGCCAC                                                                            GTAGGCGTCT                                                                            GGAACAATTT                              501   CTAAATCCCC                                                                            AATTTGGATA                                                                            GGTTGAGGTT                                                                            TACTATCTGC                                                                            TTCCTGACCA                              551   TCTACTGGCA                                                                            TAGGTTGAGA                                                                            ACGACGCAGA                                                                            AGAGCTTTAA                                                                            CACGCGCCTG                              601   CAACTCACGA                                                                            TTGGAGAAGG                                                                            GTTTCGTTAC                                                                            ATAGTCATCT                                                                            GCCCCAAGTT                              651   CCAAACCGAT                                                                            AACCTTATCA                                                                            AATTCACTAT                                                                            CTTTGGCTGA                                                                            AAGCATAAGA                              701   ATGGGCACAC                                                                            TGCTTGTCTT                                                                            ACGAATGGTC                                                                            TTAGCAACTT                                                                            CTAAACCATC                              751   AATTTCTGGA                                                                            AGCATCAAAT                                                                            CCAGAATAAT                                                                            AATATCTGGT                                                                            TGCTCTGCTT                              801   CAAATTGCTC                                                                            TAGCGCTTCA                                                                            CGACCATTAA                                                                            AAGCAGTTAC                                                                            AACTTCGTAA                              851   CCTTCCTTGG                                                                            TCATATTAAA                                                                            CTTGATAATA                                                                            TCCGAGATTG                                                                            GTTTCTCATC                              901   ATCTACAATT                                                                            AGTATTTTTA                                                                            TCATATGTTC                                                                            ACCTTTTTCT                                                                            CTACTATTAT                              951   ACCAAAAAAT                                                                            AGTCAGAAGA                                                                            CACAATAGCT                                                                            AGTCTTGGCT                                                                            ACTGTCTAAG                              1001  TTGGCTTGTG                                                                            CATAAGCCTG                                                                            CCAGATTTTT                                                                            TGTTGGGGTT                                                                            TGGCAAGTGG                              1051  GTAATTCGAT-3'                                                           (B) Response regulator polypeptide sequence deduced from the                  polynucleotide                                                                sequence in this table  SEQ ID NO: 2!.                                        NH.sub.2 -1                                                                         MIKILIVDDE                                                                            KPISDIIKFN                                                                            MTKEGYEVVT                                                                            AFNGREALEQ                                                                            FEAEQPDIII                              51    LDLMLPEIDG                                                                            LEVAKTIRKT                                                                            SSVPILMLSA                                                                            KDSEFDKVIG                                                                            LELGADDYVT                              101   KPFSNRELQA                                                                            RVKALLRRSQ                                                                            PMPVDGQEAD                                                                            SKPQPIQIGD                                                                            LEIVPDAYVA                              151   KKYGEELDLT                                                                            HREFELLYHL                                                                            ASHTGQVITR                                                                            EHLLETVNGY                                                                            DYFGDVRTVD                              201   VTVRRLREKI                                                                            EDTPSRPEYI                                                                            LTRRGVGYYM                                                                            RNNACOOH                                        (C) Polynucleotide sequence embodiments  SEQ ID NO: 1!.                       X--(R.sub.1).sub.n -1                                                               ATCGAATTAT                                                                            TGGTAATATT                                                                            GGTGATTTCA                                                                            GACCCACCTT                                                                            GCATATCAAG                              51    AACCTTGGAA                                                                            TAATCTCCTG                                                                            CAATCAAATC                                                                            TTTAACCTTT                                                                            TGATTGATTT                              101   GCTTCAACTG                                                                            AATATTATCA                                                                            CGTCTATTTC                                                                            CCAGTAATAA                                                                            GAGGGTCACA                              151   ACAAGGATGA                                                                            AACCTAACAA                                                                            AATCAGGATA                                                                            AAGATAAAAT                                                                            CTCTGGTAAA                              201   AATGGTTTGT                                                                            TTCAGTAAAT                                                                            CAAGCATTAT                                                                            TTCTCATGTA                                                                            ATACCCTACA                              251   CCACGGCGCG                                                                            TCAAGATATA                                                                            CTCTGGTCGG                                                                            CTGGGCGTAT                                                                            CTTCAATCTT                              301   CTCACGCAGA                                                                            CGTCGTACAG                                                                            TCACATCAAC                                                                            TGTACGGACA                                                                            TCACCAAAAT                              351   AGTCATAACC                                                                            CCAGACAGTC                                                                            TCAAGCAAGT                                                                            GTTCGCGCGT                                                                            GATGACTTGA                              401   CCTGTATGCG                                                                            ATGCTAAATG                                                                            ATACAAAAGC                                                                            TCAAATTCAC                                                                            GATGGGTTAA                              451   GTCTAGTTCT                                                                            TCGCCATATT                                                                            TTTTAGCCAC                                                                            GTAGGCGTCT                                                                            GGAACAATTT                              501   CTAAATCCCC                                                                            AATTTGGATA                                                                            GGTTGAGGTT                                                                            TACTATCTGC                                                                            TTCCTGACCA                              551   TCTACTGGCA                                                                            TAGGTTGAGA                                                                            ACGACGCAGA                                                                            AGAGCTTTAA                                                                            CACGCGCCTG                              601   CAACTCACGA                                                                            TTGGAGAAGG                                                                            GTTTCGTTAC                                                                            ATAGTCATCT                                                                            GCCCCAAGTT                              651   CCAAACCGAT                                                                            AACCTTATCA                                                                            AATTCACTAT                                                                            CTTTGGCTGA                                                                            AAGCATAAGA                              701   ATGGGCACAC                                                                            TGCTTGTCTT                                                                            ACGAATGGTC                                                                            TTAGCAACTT                                                                            CTAAACCATC                              751   AATTTCTGGA                                                                            AGCATCAAAT                                                                            CCAGAATAAT                                                                            AATATCTGGT                                                                            TGCTCTGCTT                              801   CAAATTGCTC                                                                            TAGCGCTTCA                                                                            CGACCATTAA                                                                            AAGCAGTTAC                                                                            AACTTCGTAA                              851   CCTTCCTTGG                                                                            TCATATTAAA                                                                            CTTGATAATA                                                                            TCCGAGATTG                                                                            GTTTCTCATC                              901   ATCTACAATT                                                                            AGTATTTTTA                                                                            TCATATGTTC                                                                            ACCTTTTTCT                                                                            CTACTATTAT                              951   ACCAAAAAAT                                                                            AGTCAGAAGA                                                                            CACAATAGCT                                                                            AGTCTTGGCT                                                                            ACTGTCTAAG                              1001  TTGGCTTGTG                                                                            CATAAGCCTG                                                                            CCAGATTTTT                                                                            TGTTGGGGTT                                                                            TGGCAAGTGG                              1051  GTAATTCGAT--(R.sub.2).sub.n --Y                                         (D) Polypeptide sequence embodiments  SEQ ID NO: 2!.                          X--(R.sub.1).sub.n -1                                                               MIKILIVDDE                                                                            KPISDIIKFN                                                                            MTKEGYEVVT                                                                            AFNGREALEQ                                                                            FEAEQPDIII                              51    LDLMLPEIDG                                                                            LEVAKTIRKT                                                                            SSVPILMLSA                                                                            KDSEFDKVIG                                                                            LELGADDYVT                              101   KPFSNRELQA                                                                            RVKALLRRSQ                                                                            PMPVDGQEAD                                                                            SKPQPIQIGD                                                                            LEIVPDAYVA                              151   KKYGEELDLT                                                                            HREFELLYHL                                                                            ASHTGQVITR                                                                            EHLLETVWGY                                                                            DYFGDVRTVD                              201   VTVRRLREKI                                                                            EDTPSRPEYI                                                                            LTRRGVGYYM                                                                            RNNA--(R.sub.2).sub.n --Y                       (E) Sequences from Streptococcus pneumoniae                                   Response regulator polynucleotide ORF sequence  SEQ ID NO: 3!.                5'-1  CGCGTGATGA                                                                            CTTGACCTGT                                                                            ATGCGATGCT                                                                            AAATGATACA                                                                            AAAGCTCAAA                              51    TTCACGATGG                                                                            GTTAAGTCTA                                                                            GTTCTTCGCC                                                                            ATATTTTTTA                                                                            GCCACGTAGG                              101   CGTCTGGAAC                                                                            AATTTCTAAA                                                                            TCCCCAATTT                                                                            GGATAGGTTG                                                                            AGGTTTACTA                              151   TCTGCTTCCT                                                                            GACCATCTAC                                                                            TGGCATAGGT                                                                            TGAGAACGAC                                                                            GCAGAAGAGC                              201   TTTAACACGC                                                                            GCCTGCAACT                                                                            CACGATTGGA                                                                            GAAGGGTTTC                                                                            GTTACATAGT                              251   CATCTGCCCC                                                                            AAGTTCCAAA                                                                            CCGATAACCT                                                                            TATCAAATTC                                                                            ACTATCTTTG                              301   GCTGAAAGCA                                                                            TAAGAATGGG                                                                            CACACTGCTT                                                                            GTCTTACGAA                                                                            TGGTCTTAGC                              351   AACTTCTAAA                                                                            CCATCAATTT                                                                            CTGGAAGCAT                                                                            CAAATCCAGA                                                                            ATAATAATAT                              401   CTGGTTGCTC                                                                            TGCTTCAAAT                                                                            TGCTCTAGCG                                                                            CTTCACGAAC                                                                            ATTAAAAGCA                              451   GTTACAACTT                                                                            CGTAACCTTC                                                                            CTTGGTCATA                                                                            TTAAAACTTG                                                                            ATAATATCCG                              501   AGATTGGTTC                                                                            TCATCATCTA                                                                            CAATTAGTAT                                                                            TTT-3'                                          (F) Response regulator polypeptide sequence deduced from the                  polynucleotide ORF                                                            sequence in this table  SEQ ID NO: 4!.                                        NH.sub.2 -1                                                                         KILIVDDENQ                                                                            SRILSSFNMT                                                                            KEGYEWTAF                                                                             NVREALEQFE                                                                            AEQPDIIILD                              51    LMLPEIDGLE                                                                            VAKTIRKTSS                                                                            VPILMLSAKD                                                                            SEFDKVIGLE                                                                            LGADDYVTKP                              101   FSNRELQARV                                                                            KALLRRSQPM                                                                            PVDGQEADSK                                                                            PQPIQIGDLE                                                                            IVPDAYVAKK                              151   YGEELDLTHR                                                                            EFELLYHLAS                                                                            HTGQVIT--COOH                                           __________________________________________________________________________

Deposited Materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on 11 Apr. 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit. On 17 Apr.1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli wassimilarly deposited with the NCIMB and assigned deposit number 40800.The Streptococcus pneumoniae strain deposit is referred to herein as"the deposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length Response regulator gene.The sequence of the polynucleotides contained in the deposited strain,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1 SEQID NO:2! (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity ofResponse regulator, and also those which have at least 70% identity to apolypeptide of Table 1 SEQ ID NOS:2 and 4! or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 SEQ IDNOS:2 and 4!, and more preferably at least 90% similarity (morepreferably at least 90% identity) to a polypeptide of Table 1 SEQ IDNOS:2 and 4! and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to a polypeptide of Table 1 SEQID NOS:2 and 4! and also include portions of such polypeptides with suchportion of the polypeptide generally containing at least 30 amino acidsand more preferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) SEQ ID NO:2! wherein, at the amino terminus, X is hydrogen,and at the carboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is anyamino acid residue, and n is an integer between 1 and 1000. Any stretchof amino acid residues denoted by either R group, where R is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with Response regulator polypeptidesfragments may be "free-standing," or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 SEQ ID NOS:2 and 4!, orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Streptococcus pneumoniae,are also preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of Response regulator, including thosewith a similar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the Response regulatorpolypeptide having a deduced amino acid sequence of Table 1 SEQ ID NOS:2and 4! and polynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 SEQ ID NOS:1 and 3!, a polynucleotide of theinvention encoding Response regulator polypeptide may be obtained usingstandard cloning and screening methods, such as those for cloning andsequencing chromosomal DNA fragments from bacteria using Streptococcuspneumoniae 0100993 cells as starting material, followed by obtaining afull length clone. For example, to obtain a polynucleotide sequence ofthe invention, such as a sequence given in Table 1 SEQ ID NOS:1 and 3!,typically a library of clones of chromosomal DNA of Streptococcuspneumoniae 0100993 in E.coli or some other suitable host is probed witha radiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 SEQ ID NO:1! was discovered in a DNA library derived fromStreptococcus pneumoniae 0100993.

The DNA sequence set out in Table 1 SEQ ID NOS:1! contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 SEQ ID NOS:2! with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The start codon of the DNA in Table 1 isnucleotide number 137 and last codon that encodes an amino acid isnumber 838, the stop codon being the next codon following this lastcodon encoding an amino acid.

Response regulator of the invention is structurally related to otherproteins of the Response regulator family, as shown by the results ofsequencing the DNA encoding Response regulator of the deposited strain.The protein exhibits greatest homology to Bacillus subtilis YYCF proteinamong known proteins. Response regulator of Table 1 SEQ ID NO:2! hasabout 67% identity over its entire length and about 80% similarity overits entire length with the amino acid sequence of Bacillus subtilis YYCFpolypeptide.

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 SEQ ID NO:1!. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., U.S.A. 86: 821-824 (1989), or an HA tag (Wilson etal., Cell 37: 767 (1984). Polynucleotides of the invention also include,but are not limited to, polynucleotides comprising a structural gene andits naturally associated sequences that control gene expression.

A preferred embodiment of the invention is the polynucleotide ofcomprising nucleotide 137 to 838 set forth in SEQ ID NO:1 of Table 1which encodes the Response regulator polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C) SEQ ID NO:1! wherein, at the 5' end of the molecule, X ishydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal,R₁ and R₂ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneunoniae Responseregulator having the amino acid sequence set out in Table 1 SEQ IDNO:2!. The term also encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, interrupted by integrated phage or an insertion sequence orediting) together with additional regions, that also may contain codingand/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 SEQ ID NO:2!. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingResponse regulator variants, that have the amino acid sequence ofResponse regulator polypeptide of Table 1 SEQ ID NO:2! in which several,a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, that donot alter the properties and activities of Response regulator.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding Response regulator polypeptide having an amino acid sequenceset out in Table 1 SEQ ID NOS:2 and 4!, and polynucleotides that arecomplementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least80% identical over its entire length to a polynucleotide encodingResponse regulator polypeptide of the deposited strain andpolynucleotides complementary thereto. In this regard, polynucleotidesat least 90% identical over their entire length to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 SEQ ID NO:1!.

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 or SEQ ID NO:3 under stringent hybridization conditions witha probe having the sequence of said polynucleotide sequence set forth inSEQ ID NO:1 or a fragment thereof; and isolating said DNA sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding Responseregulator and to isolate cDNA and genomic clones of other genes thathave a high sequence similarity to the Response regulator gene. Suchprobes generally will comprise at least 15 bases. Preferably, suchprobes will have at least 30 bases and may have at least 50 bases.Particularly preferred probes will have at least 30 bases and will have50 bases or less.

For example, the coding region of the Response regulator gene may beisolated by screening using the known DNA sequence provided in SEQ IDNO: 1 to synthesize an oligonucleotide probe. A labeled oligonucleotidehaving a sequence complementary to that of a gene of the invention isthen used to screen a library of cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the Response regulatorpolynucleotides of the invention for use as diagnostic reagents.Detection of Response regulator in a eukaryote, particularly a mammal,and especially a human, will provide a diagnostic method for diagnosisof a disease. Eukaryotes (herein also "individual(s)"), particularlymammals, and especially humans, infected with an organism comprising theResponse regulator gene may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled Response regulator polynucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations in theelectrophoretic mobility of the DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,Science, 230: 1242 (1985). Sequence changes at specific locations alsomay be revealed by nuclease protection assays, such as RNase and S 1protection or a chemical cleavage method. See, e.g., Cotton et al.,Proc. Natl. Acad. Sci., U.S.A., 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding Response regulator can be used to identify and analyzemutations. Examples of representative primers are shown below in Table2.

                  TABLE 2                                                         ______________________________________                                        Primers for amplification of Response regulator polynucleotides               SEQ ID NO                                                                              PRIMER SEQUENCE                                                      ______________________________________                                        5        5'- ATGATAAAAATACTAATTGTAGATGATG-3'                                  6        5'- AGCATTATTTCTCATGTAATACCCTACA-3'                                  ______________________________________                                    

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying Response regulator DNA isolatedfrom a sample derived from an individual. The primers may be used toamplify the gene isolated from an infected individual such that the genemay then be subject to various techniques for elucidation of the DNAsequence. In this way, mutations in the DNA sequence may be detected andused to diagnose infection and to serotype and/or classify theinfectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, and most preferably otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, comprising determining from asample derived from an individual a increased level of expression ofpolynucleotide having the sequence of Table 1 SEQ ID NO:1!. Increased ordecreased expression of Response regulator polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting and other hybridizationmethods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of Response regulator protein compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a Response regulator protein, in a sample derived from a hostare well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Response regulator or from naive libraries(McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al.,(1992) Biotechnology 10, 779-783). The affinity of these antibodies canalso be improved by chain shuffling (Clackson, T. et al., (1991) Nature352, 624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against Response regulator- polypeptidemay be employed to treat infections, particularly bacterial infectionsand especially otitis media, conjunctivitis, pneumonia, bacteremia,meningitis, sinusitis, pleural empyema and endocarditis, and mostparticularly meningitis, such as for example infection of cerebrospinalfluid.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,( 1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS, 1986:83,9551), encapsulation of DNA in various forms ofliposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS 1984:81,5849).

Antagonists and Agonists--Assays and Molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (199 1).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action ofResponse regulator polypeptides or polynucleotides, particularly thosecompounds that are bacteriostatic and/or bacteriocidal. The method ofscreening may involve high-throughput techniques. For example, to screenfor agonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising Response regulator polypeptideand a labeled substrate or ligand of such polypeptide is incubated inthe absence or the presence of a candidate molecule that may be aResponse regulator agonist or antagonist. The ability of the candidatemolecule to agonize or antagonize the Response regulator polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of Response regulatorpolypeptide are most likely to be good antagonists. Molecules that bindwell and increase the rate of product production from substrate areagonists. Detection of the rate or level of production of product fromsubstrate may be enhanced by using a reporter system. Reporter systemsthat may be useful in this regard include but are not limited tocolorimetric labeled substrate converted into product, a reporter genethat is responsive to changes in Response regulator polynucleotide orpolypeptide activity, and binding assays known in the art.

Another example of an assay for Response regulator antagonists is acompetitive assay that combines Response regulator and a potentialantagonist with Response regulator-binding molecules, recombinantResponse regulator binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. Response regulator can be labeled, such asby radioactivity or a colorimetric compound, such that the number ofResponse regulator molecules bound to a binding molecule or converted toproduct can be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing Response regulator-induced activities, thereby preventing theaction of Response regulator by excluding Response regulator frombinding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of Response regulator.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock Response regulator protein-mediated mammalian cell invasion by,for example, initiating phosphorylation of mammalian tyrosine kinases(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block bacterialadhesion between mammalian extracellular matrix proteins and bacterialResponse regulator proteins that mediate tissue damage and; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with Response regulator, or afragment or variant thereof, adequate to produce antibody and/or T cellimmune response to protect said individual from infection, particularlybacterial infection and most particularly Streptococcus pneumoniaeinfection. Also provided are methods whereby such immunological responseslows bacterial replication. Yet another aspect of the invention relatesto a method of inducing immunological response in an individual whichcomprises delivering to such individual a nucleic acid vector to directexpression of Response regulator, or a fragment or a variant thereof,for expressing Response regulator, or a fragment or a variant thereof invivo in order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual from disease, whether that disease is already establishedwithin the individual or not. One way of administering the gene is byaccelerating it into the desired cells as a coating on particles orotherwise. Such nucleic acid vector may comprise DNA, RNA, a modifiednucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a Response regulator or protein codedtherefrom, wherein the composition comprises a recombinant Responseregulator or protein coded therefrom comprising DNA which codes for andexpresses an antigen of said Response regulator or protein codedtherefrom. The immunological response may be used therapeutically orprophylactically and may take the form of antibody immunity or cellularimmunity such as that arising from CTL or CD4+ T cells.

A Response regulator polypeptide or a fragment thereof may be fused withco-protein which may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352(1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certainResponse regulator protein, it is to be understood that this coversfragments of the naturally occurring protein and similar proteins withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant protein.

Compositions, Kits and Administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Streptococcuspneumoniae in E. coli. The sequencing data from two or more clonescontaining overlapping Streptococcus pneumoniae DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumnoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 1--1 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E.coli infected with the packagedlibrary. The library is amplified by standard procedures.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1060 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATCGAATTATTGGTAATATTGGTGATTTCAGACCCACCTTGCATATCAAGAACCTTGGAA60                TAATCTCCTGCAATCAAATCTTTAACCTTTTGATTGATTTGCTTCAACTGAATATTATCA120               CGTCTATTTCCCAGTAATAAGAGGGTCACAACAAGGATGAAACCTAACAAAATCAGGATA180               AAGATAAAATCTCTGGTAAAAATGGTTTGTTTCAGTAAATCAAGCATTATTTCTCATGTA240               ATACCCTACACCACGGCGCGTCAAGATATACTCTGGTCGGCTGGGCGTATCTTCAATCTT300               CTCACGCAGACGTCGTACAGTCACATCAACTGTACGGACATCACCAAAATAGTCATAACC360               CCAGACAGTCTCAAGCAAGTGTTCGCGCGTGATGACTTGACCTGTATGCGATGCTAAATG420               ATACAAAAGCTCAAATTCACGATGGGTTAAGTCTAGTTCTTCGCCATATTTTTTAGCCAC480               GTAGGCGTCTGGAACAATTTCTAAATCCCCAATTTGGATAGGTTGAGGTTTACTATCTGC540               TTCCTGACCATCTACTGGCATAGGTTGAGAACGACGCAGAAGAGCTTTAACACGCGCCTG600               CAACTCACGATTGGAGAAGGGTTTCGTTACATAGTCATCTGCCCCAAGTTCCAAACCGAT660               AACCTTATCAAATTCACTATCTTTGGCTGAAAGCATAAGAATGGGCACACTGCTTGTCTT720               ACGAATGGTCTTAGCAACTTCTAAACCATCAATTTCTGGAAGCATCAAATCCAGAATAAT780               AATATCTGGTTGCTCTGCTTCAAATTGCTCTAGCGCTTCACGACCATTAAAAGCAGTTAC840               AACTTCGTAACCTTCCTTGGTCATATTAAACTTGATAATATCCGAGATTGGTTTCTCATC900               ATCTACAATTAGTATTTTTATCATATGTTCACCTTTTTCTCTACTATTATACCAAAAAAT960               AGTCAGAAGACACAATAGCTAGTCTTGGCTACTGTCTAAGTTGGCTTGTGCATAAGCCTG1020              CCAGATTTTTTGTTGGGGTTTGGCAAGTGGGTAATTCGAT1060                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 234 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetIleLysIleLeuIleValAspAspGluLysProIleSerAspIle                              151015                                                                        IleLysPheAsnMetThrLysGluGlyTyrGluValValThrAlaPhe                              202530                                                                        AsnGlyArgGluAlaLeuGluGlnPheGluAlaGluGlnProAspIle                              354045                                                                        IleIleLeuAspLeuMetLeuProGluIleAspGlyLeuGluValAla                              505560                                                                        LysThrIleArgLysThrSerSerValProIleLeuMetLeuSerAla                              65707580                                                                      LysAspSerGluPheAspLysValIleGlyLeuGluLeuGlyAlaAsp                              859095                                                                        AspTyrValThrLysProPheSerAsnArgGluLeuGlnAlaArgVal                              100105110                                                                     LysAlaLeuLeuArgArgSerGlnProMetProValAspGlyGlnGlu                              115120125                                                                     AlaAspSerLysProGlnProIleGlnIleGlyAspLeuGluIleVal                              130135140                                                                     ProAspAlaTyrValAlaLysLysTyrGlyGluGluLeuAspLeuThr                              145150155160                                                                  HisArgGluPheGluLeuLeuTyrHisLeuAlaSerHisThrGlyGln                              165170175                                                                     ValIleThrArgGluHisLeuLeuGluThrValTrpGlyTyrAspTyr                              180185190                                                                     PheGlyAspValArgThrValAspValThrValArgArgLeuArgGlu                              195200205                                                                     LysIleGluAspThrProSerArgProGluTyrIleLeuThrArgArg                              210215220                                                                     GlyValGlyTyrTyrMetArgAsnAsnAla                                                225230                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 533 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGCGTGATGACTTGACCTGTATGCGATGCTAAATGATACAAAAGCTCAAATTCACGATGG60                GTTAAGTCTAGTTCTTCGCCATATTTTTTAGCCACGTAGGCGTCTGGAACAATTTCTAAA120               TCCCCAATTTGGATAGGTTGAGGTTTACTATCTGCTTCCTGACCATCTACTGGCATAGGT180               TGAGAACGACGCAGAAGAGCTTTAACACGCGCCTGCAACTCACGATTGGAGAAGGGTTTC240               GTTACATAGTCATCTGCCCCAAGTTCCAAACCGATAACCTTATCAAATTCACTATCTTTG300               GCTGAAAGCATAAGAATGGGCACACTGCTTGTCTTACGAATGGTCTTAGCAACTTCTAAA360               CCATCAATTTCTGGAAGCATCAAATCCAGAATAATAATATCTGGTTGCTCTGCTTCAAAT420               TGCTCTAGCGCTTCACGAACATTAAAAGCAGTTACAACTTCGTAACCTTCCTTGGTCATA480               TTAAAACTTGATAATATCCGAGATTGGTTCTCATCATCTACAATTAGTATTTT533                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 177 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LysIleLeuIleValAspAspGluAsnGlnSerArgIleLeuSerSer                              151015                                                                        PheAsnMetThrLysGluGlyTyrGluValValThrAlaPheAsnVal                              202530                                                                        ArgGluAlaLeuGluGlnPheGluAlaGluGlnProAspIleIleIle                              354045                                                                        LeuAspLeuMetLeuProGluIleAspGlyLeuGluValAlaLysThr                              505560                                                                        IleArgLysThrSerSerValProIleLeuMetLeuSerAlaLysAsp                              65707580                                                                      SerGluPheAspLysValIleGlyLeuGluLeuGlyAlaAspAspTyr                              859095                                                                        ValThrLysProPheSerAsnArgGluLeuGlnAlaArgValLysAla                              100105110                                                                     LeuLeuArgArgSerGlnProMetProValAspGlyGlnGluAlaAsp                              115120125                                                                     SerLysProGlnProIleGlnIleGlyAspLeuGluIleValProAsp                              130135140                                                                     AlaTyrValAlaLysLysTyrGlyGluGluLeuAspLeuThrHisArg                              145150155160                                                                  GluPheGluLeuLeuTyrHisLeuAlaSerHisThrGlyGlnValIle                              165170175                                                                     Thr                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATGATAAAAATACTAATTGTAGATGATG28                                                (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGCATTATTTCTCATGTAATACCCTACA28                                                __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising a polynucleotide sequence encoding a response regulator polypeptide having an amino acid sequence that is at least 80% identical over its entire length to the amino acid sequence set forth in SEQ ID NO:2,whereby said encoded amino acid sequence is identical to SEQ ID NO:2 except that it may include up to 20 amino acid alterations per each 100 amino acids of SEQ ID NO:2, wherein said alterations are selected from the group consisting of amino acid deletions, substitutions, or insertions, and wherein said alterations may occur at the amino terminus or the carboxyl terminus of SEQ ID NO:2 or may occur anywhere between said termini, interspersed either individually among the amino acid sequence of SEQ ID NO:2 or in one or more contiguous groups within the amino acid sequence of SEQ ID NO:2.
 2. The isolated polynucleotide of claim 1 comprising a polynucleotide sequence encoding a response regulator polypeptide having an amino acid sequence that is at least 80% identical over its entire length to a polynucleotide encoding the same mature polypeptide expressed by the response regulator gene contained in the Streptococcus pneumoniae strain deposited with the accession number NCIMB 40794,whereby said encoding polynucleotide sequence is identical to the polynucleotide sequence encoding the response regulator of said deposited strain except that it may include up to 20 point mutations per each 100 nucleotides of the polynucleotide sequence encoding the response regulator of said deposited strain, wherein said point mutations are selected from the group consisting of nucleotide deletions, substitutions, or insertions, and wherein said alterations may occur at the 5'-terminus or the 3'-terminus of the polynucleotide sequence encoding the response regulator of said deposited strain or may occur anywhere between said termini, interspersed either individually among the nucleotide sequence of the polynucleotide sequence encoding the response regulator of said deposited strain or in one or more contiguous groups within the nucleotide sequence of the polynucleotide sequence encoding the response regulator of said deposited strain.
 3. The polynucleotide of claim 1 wherein said polynucleotide sequence encoding a response regulator polypeptide is DNA or RNA.
 4. The polynucleotide of claim 1 wherein said polynucleotide sequence encoding a response regulator polypeptide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
 5. The polynucleotide of claim 1 wherein said polynucleotide sequence encoding a response regulator polypeptide consists of the nucleic acid sequence set forth in SEQ ID NO:1.
 6. The polynucleotide of claim 5 wherein said polynucleotide sequence encoding a response regulator polypeptide consists of the nucleotide sequence from position 137 to position 838, inclusive, of the polynucleotide sequence set forth in SEQ ID NO:1.
 7. The polynucleotide of claim 1 wherein said polynucleotide sequence encoding a response regulator polypeptide consists of the structure of:

    __________________________________________________________________________     X--(R.sub.1).sub.n -1                                                                ATCGAATTAT                                                                             TGGTAATATT                                                                             GGTGATTTCA                                                                             GACCCACCTT                                                                             GCATATCAAG                               51    AACCTTGGAA                                                                             TAATCTCCTG                                                                             CAATCAAATC                                                                             TTTAACCTTT                                                                             TGATTGATTT                               101   GCTTCAACTG                                                                             AATATTATCA                                                                             CGTCTATTTC                                                                             CCAGTAATAA                                                                             GAGGGTCACA                               151   ACAAGGATGA                                                                             AACCTAACAA                                                                             AATCAGGATA                                                                             AAGATAAAAT                                                                             CTCTGGTAAA                               201   AATGGTTTGT                                                                             TTCAGTAAAT                                                                             CAAGCATTAT                                                                             TTCTCATGTA                                                                             ATACCCTACA                               251   CCACGGCGCG                                                                             TCAAGATATA                                                                             CTCTGGTCGG                                                                             CTGGGCGTAT                                                                             CTTCAATCTT                               301   CTCACGCAGA                                                                             CGTCGTACAG                                                                             TCACATCAAC                                                                             TGTACGGACA                                                                             TCACCAAAAT                               351   AGTCATAACC                                                                             CCAGACAGTC                                                                             TCAAGCAAGT                                                                             GTTCGCGCGT                                                                             GATGACTTGA                               401   CCTGTATGCG                                                                             ATGCTAAATG                                                                             ATACAAAAGC                                                                             TCAAATTCAC                                                                             GATGGGTTAA                               451   GTCTAGTTCT                                                                             TCGCCATATT                                                                             TTTTAGCCAC                                                                             GTAGGCGTCT                                                                             GGAACAATTT                               501   CTAAATCCCC                                                                             AATTTGGATA                                                                             GGTTGAGGTT                                                                             TACTATCTGC                                                                             TTCCTGACCA                               551   TCTACTGGCA                                                                             TAGGTTGAGA                                                                             ACGACGCAGA                                                                             AGAGCTTTAA                                                                             CACGCGCCTG                               601   CAACTCACGA                                                                             TTGGAGAAGG                                                                             GTTTCGTTAC                                                                             ATAGTCATCT                                                                             GCCCCAAGTT                               651   CCAAACCGAT                                                                             AACCTTATCA                                                                             AATTCACTAT                                                                             CTTTGGCTGA                                                                             AAGCATAAGA                               701   ATGGGCACAC                                                                             TGCTTGTCTT                                                                             ACGAATGGTC                                                                             TTAGCAACTT                                                                             CTAAACCATC                               751   AATTTCTGGA                                                                             AGCATCAAAT                                                                             CCAGAATAAT                                                                             AATATCTGGT                                                                             TGCTCTGCTT                               801   CAAATTGCTC                                                                             TAGCGCTTCA                                                                             CGACCATTAA                                                                             AAGCAGTTAC                                                                             AACTTCGTAA                               851   CCTTCCTTGG                                                                             TCATATTAAA                                                                             CTTGATAATA                                                                             TCCGAGATTG                                                                             GTTTCTCATC                               901   ATCTACAATT                                                                             AGTATTTTTA                                                                             TCATATGTTC                                                                             ACCTTTTTCT                                                                             CTACTATTAT                               951   ACCAAAAAAT                                                                             AGTCAGAAGA                                                                             CACAATAGCT                                                                             AGTCTTGGCT                                                                             ACTGTCTAAG                               1001  TTGGCTTGTG                                                                             CATAAGCCTG                                                                             CCAGATTTTT                                                                             TGTTGGGGTT                                                                             TGGCAAGTGG                               1051  GTAATTCGAT--*r.sub.2).sub.N--y                                           __________________________________________________________________________

wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal, R₁ and R₂ is any nulceic acid residue, and n is an integer between 1 and
 1000. 8. An isolated polynucleotide comprising a polynucleotide sequence encoding a response regulator polypeptide having an amino acid sequence that is at least 90% identical over its entire length to the amino acid sequence set forth in SEQ ID NO:2,whereby said encoded amino acid sequence is identical to SEQ ID NO:2 except that it may include up to 10 amino acid alterations per each 100 amino acids of SEQ ID NO:2, wherein said alterations are selected from the group consisting of amino acid deletions, substitutions, or insertions, and wherein said alterations may occur at the amino terminus or the carboxyl terminus of SEQ ID NO:2 or may occur anywhere between said termini, interspersed either individually among the amino acid sequence of SEQ ID NO:2 or in one or more contiguous groups within the amino acid sequence of SEQ ID NO:2.
 9. The isolated polynucleotide of claim 8 comprising a polynucleotide sequence encoding a response regulator polypeptide having an amino acid sequence that is at least 90% identical over its entire length to a polynucleotide encoding the same mature polypeptide expressed by the response regulator gene contained in the Streptococcus pneumoniae strain deposited with the accession number NCIMB 40794,whereby said encoding polynucleotide sequence is identical to the polynucleotide sequence encoding the response regulator of said deposited strain except that it may include up to 10 point mutations per each 100 nucleotides of the polynucleotide sequence encoding the response regulator of said deposited strain, wherein said point mutations are selected from the group consisting of nucleotide deletions, substitutions, or insertions, and wherein said alterations may occur at the 5'-terminus or the 3'-terminus of the polynucleotide sequence encoding the response regulator of said deposited strain or may occur anywhere between said termini, interspersed either individually among the nucleotide sequence of the polynucleotide sequence encoding the response regulator of said deposited strain or in one or more contiguous groups within the nucleotide sequence of the polynucleotide sequence encoding the response regulator of said deposited strain.
 10. An isolated polynucleotide comprising a polynucleotide sequence encoding a response regulator polypeptide having an amino acid sequence that is at least 95% identical over its entire length to the amino acid sequence set forth in SEQ ID NO:2,whereby said encoded amino acid sequence is identical to SEQ ID NO:2 except that it may include up to 5 amino acid alterations per each 100 amino acids of SEQ ID NO:2, wherein said alterations are selected from the group consisting of amino acid deletions, substitutions, or insertions, and wherein said alterations may occur at the amino terminus or the carboxyl terminus of SEQ ID NO:2 or may occur anywhere between said termini, interspersed either individually among the amino acid sequence of SEQ ID NO:2 or in one or more contiguous groups within the amino acid sequence of SEQ ID NO:2.
 11. The polynucleotide of claim 10 wherein said polynucleotide sequence encoding a response regulator polypeptide is DNA or RNA.
 12. The isolated polynucleotide of claim 10 comprising a polynucleotide sequence encoding a response regulator polypeptide having an amino acid sequence that is at least 95% identical over its entire length to a polynucleotide encoding the same mature polypeptide expressed by the response regulator gene contained in the Streptococcus pneumoniae strain deposited with the accession number NCIMB 40794,whereby said encoding polynucleotide sequence is identical to the polynucleotide sequence encoding the response regulator of said deposited strain except that it may include up to 5 point mutations per each 100 nucleotides of the polynucleotide sequence encoding the response regulator of said deposited strain, wherein said point mutations are selected from the group consisting of nucleotide deletions, substitutions, or insertions, and wherein said alterations may occur at the 5'-terminus or the 3'-terminus of the polynucleotide sequence encoding the response regulator of said deposited strain or may occur anywhere between said termini, interspersed either individually among the nucleotide sequence of the polynucleotide sequence encoding the response regulator of said deposited strain or in one or more contiguous groups within the nucleotide sequence of the polynucleotide sequence encoding the response regulator of said deposited strain.
 13. An isolated polynucleotide comprising a polynucleotide sequence encoding a response regulator polypeptide having the amino acid sequence set forth in SEQ ID NO:2.
 14. An polynucleotide encoding a fusion polypeptide that is at least 80% identical over its entire length to the amino acid sequence set forth in SEQ ID NO:2 wherein said fusion polypeptide comprises the amino acid sequence of SEQ ID NO:2,whereby the amino acid sequence of said fusion polypeptide is identical to SEQ ID NO:2 except that it may include up to 20 amino acid alterations per each 100 amino acids of SEQ ID NO:2, wherein said alterations are amino acid insertions occurring at either the amino terminus or the carboxyl terminus of the amino acid sequence of SEQ ID NO:2.
 15. An isolated polynucleotide comprising a polynucleotide sequence encoding the same mature polypeptide expressed by the response regulator gene contained in the Streptococcus pneumoniae strain deposited with the accession number NCIMB
 40794. 16. A polynucleotide comprising the nucleotide sequence from position 137 to position 838, inclusive, of the polynucleotide sequence set forth in SEQ ID NO:1.
 17. An isolated polynucleotide comprising a polynucleotide sequence encoding a response regulator polypeptide that is at least 80% identical over its entire length to the amino acid sequence set forth in SEQ ID NO:4,whereby said encoded amino acid sequence is identical to SEQ ID NO:4 except that it may include up to 20 amino acid alterations per each 100 amino acids of SEQ ID NO:4, wherein said alterations are selected from the group consisting of amino acid deletions, substitutions, or insertions, and wherein said alterations may occur at the amino terminus or the carboxyl terminus of SEQ ID NO:4 or may occur anywhere between said termini, interspersed either individually among the amino acid sequence of SEQ ID NO:4 or in one or more contiguous groups within the amino acid sequence of SEQ ID NO:4.
 18. An isolated polynucleotide comprising a polynucleotide sequence encoding a response regulator polypeptide that is at least 95% identical over its entire length to the amino acid sequence set forth in SEQ ID NO:4,whereby said encoded amino acid sequence is identical to SEQ ID NO:4 except that it may include up to 5 amino acid alterations per each 100 amino acids of SEQ ID NO:4, wherein said alterations are selected from the group consisting of amino acid deletions, substitutions, or insertions, and wherein said alterations may occur at the amino terminus or the carboxyl terminus of SEQ ID NO:4 or may occur anywhere between said termini, interspersed either individually among the amino acid sequence of SEQ ID NO:4 or in one or more contiguous groups within the amino acid sequence of SEQ ID NO:4.
 19. An isolated polynucleotide comprising a polynucleotide sequence encoding a response regulator polypeptide having the amino acid sequence set forth in SEQ ID NO:4.
 20. The polynucleotide of any of claims 5, 17, 18 or 19 wherein said polynucleotide sequence encoding a response regulator polypeptide consists of the polynucleotide sequence of SEQ ID NO:3.
 21. The polynucleotide of claim 1, 8, 9, 10, 12, 13, 15 or 24, wherein said polynucleotide encodes a response regulator polypeptide that is bound specifically by a histidine kinase of Sireptcoccus.
 22. A polynucleotide which is complementary to a polynucleotide of any of claims 1, 8, 15, 24, 25, 14, 17, 18 and
 19. 23. An isolated polynucleotide comprising a polynucleotide sequence that is at least 80% identical over its entire length to a polynucleotide encoding the polypeptide sequence set forth in SEQ ID NO:2 and which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:1,whereby said polynucleotide sequence is identical to SEQ ID NO:1 except that it may include up to 20 point mutations per each 100 nucleotides of SEQ ID NO:1, wherein said mutations are selected from the group consisting of nucleotide deletions, substitutions, or insertions, and wherein said alterations may occur at the 5'-terminus or the 3'-terminus of the polynucleotide sequence or may occur anywhere between said termini, interspersed either individually among the nucleotide sequence or in one or more contiguous groups within the nucleotide sequence.
 24. An isolated polynucleotide comprising a polynucleotide sequence that is at least 80% identical over its entire length to the polynucleotide sequence set forth in SEQ ID NO:1 and which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:1,whereby said polynucleotide sequence is identical to SEQ ID NO:1 except that it may include up to 20 point mutations per each 100 nucleotides of SEQ ID NO:1, wherein said mutations are selected from the group consisting of nucleotide deletions, substitutions, or insertions, and wherein said alterations may occur at the 5'-terminus or the 3'-terminus of the polynucleotide sequence or may occur anywhere between said termini, interspersed either individually among the nucleotide sequence or in one or more contiguous groups within the nucleotide sequence.
 25. An isolated polynucleotide comprising a polynucleotide sequence that is at least 90% identical over its entire length to a polynucleotide encoding the polypeptide sequence set forth in SEQ ID NO:2 and which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:1,whereby said polynucleotide sequence is identical to SEQ ID NO:1 except that it may include up to 10 point mutations per each 100 nucleotides of SEQ ID NO:1, wherein said mutations are selected from the group consisting of nucleotide deletions, substitutions, or insertions, and wherein said alterations may occur at the 5'-terminus or the 3'-terminus of the polynucleotide sequence or may occur anywhere between said termini, interspersed either individually among the nucleotide sequence or in one or more contiguous groups within the nucleotide sequence.
 26. An isolated polynucleotide comprising a polynucleotide sequence that is at least 90% identical over its entire length to the polynucleotide sequence set forth in SEQ ID NO:1 and which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:1,whereby said polynucleotide sequence is identical to SEQ ID NO:1 except that it may include up to 10 point mutations per each 100 nucleotides of SEQ ID NO:1, wherein said mutations are selected from the group consisting of nucleotide deletions, substitutions, or insertions, and wherein said alterations may occur at the 5'-terminus or the 3'-terminus of the polynucleotide sequence or may occur anywhere between said termini, interspersed either individually among the nucleotide sequence or in one or more contiguous groups within the nucleotide sequence.
 27. A vector comprising the polynucleotide of claim
 3. 28. A vector comprising the polynucleotide of claim
 11. 29. A host cell comprising the vector of claim
 27. 30. A host cell comprising the vector of claim
 28. 31. A process for producing a response regulator polypeptide comprising the step of culturing the host of claim 29 under conditions sufficient for the production of said polypeptide.
 32. A process for producing a response regulator polypeptide comprising the step of culturing a host of claim 30 under conditions sufficient for the production of said polypeptide. 